The present invention relates to novel methods for the isolation and culture of vasculogenic progenitor cells from stem cells and, more particularly, to methods for use of vasculogenic progenitor cells in tissue engineering, research and diagnostics.
Recently, techniques have been developed which allow human embryonic stem cells to proliferate indefinitely in culture, enabling experimentation with induction of differentiation in a directed, tissue-specific manner (Itskovitz-Eldor, J et al Mol Med 2000; 6:88-95, Reubinoff B E at al Nat Biotech 2000; 18:399-404, Schuldiner M et al PNAS USA 2000; 97:11307-12). Human embryonic stem cell growth and development is being carefully studied, and the rapidly accumulating knowledge is being employed in a variety of innovative therapeutic applications including in-vitro tissue engineering, transplantation medicine, generation of transgenic embryos and treatment of degenerative disease. Most significantly, the President of the U.S. has recognized the overwhelming importance of embryonic stem cells to medicine and research, and has recently sanctioned projects using existing human embryonic stem cell lines (White House Fact Sheet: Embryonic Stem Cell Research, Aug. 9, 2001). However, in-vitro manipulation of the complex steps of development, to reliably produce substantial amounts of desired cell lineages and specific phenotypes remains a crucially important goal.
Blood Vessel Formation in Embryonic Development and Adult Life
In the early stages of embryonic development, vessel formation occurs by a process referred to as vasculogenesis, in which mesodermally-derived endothelial cell progenitors undergo de-novo differentiation, expand and coalescence to form a network of primitive tubules (Yancopoulos G D et al Nature 2000; 407:242). These blood vessels are generally composed of two cell lineages, each serving a different function: internal endothelial cells that form the channels for blood conduction, but alone cannot complete vasculogenesis; and periendothelial smooth muscle cells that protect and stabilize the fragile channels from rupture and provide haemostatic control (Carmeliet P Nature Med 2000; 6:389). A third cell lineage, the hematopoietic cells, share a common progenitor with the vascular cells, and differentiate into the blood cells. In the vertebrate embryo vasculogenesis occurs in the paraxial and lateral mesoderm, giving rise to the primordia of the heart, the dorsal aorta, and large vessels of the head, lung and gastrointestinal system. Angiogenesis involves the maturation and remodeling of the primitive vascular plexus into a complex network of large and small vessels. Angiogenesis also leads to vascularization of initially avascular organs such as kidney, brain and limb buds.
Angiogenesis is also required postnatally for normal tissue growth, and continues throughout adult life, for example during neo-vascularization of the endometrium during normal female estrus, during pregnancy in the placenta, and during wound healing (Risau, et al Nature 1997; 386:671-674).
In addition, a number of diseases and disorders have been associated with abnormal endothelial growth: endothelial hyperproliferation in atherosclerosis, neovascularization in tumor growth and metastasis, and deregulated angiogenesis in rheumatoid arthritis, retinopathies, hemangiomas and psoriasis (Folkman et al Nature Med. 1995; 1: 27-31; Hanahan and Folkman, Cell 1996; 86:353-64).
Embryonic Endothelial Cells In-vitro
Research into the functions, origin and nature of embryonic endothelial cells (EEC) has revealed that EECs can promote liver organogenesis (Matsumoto K et al Science 2001; 294:559), induce pancreas differentiation (Lammert E et al 2001; 294:564) and trans-differentiate into cardiac muscle cells under specific conditions (Condorelli G et al 2001; 98:10733). While the nature of differentiation and development of endothelial precursors is not yet fully understood, it is becoming clear that hematopoietic development and the generation of vascular smooth muscle cells (v-SMC) are tightly linked with vascular development.
Embryonic stem cells are difficult to maintain in culture, tending to spontaneously differentiate. For ongoing cultures, cells from the inner mass of blastocysts are typically grown on a layer of mouse embryonic fibroblast “feeder” cells to preserve their undifferentiated phenotype and proliferabilty (Keller, G M Curr Opin Cell Biol 1995; 7:862-69). In mice, early differentiation into embryonically distinct cell types can be induced by coculture with stromal cell lines (Palacios R, et al PNAS USA 1995; 92:7530-34), culture on substrates such as fibronectin, laminin, collagen, etc. (Ogawa M et al Blood 1999; 93: 1168-77) or in vitro aggregation of embryoid stem (ES) cells into “embryoid bodies” (EB), demonstrating regional differentiation into three germ layers (Keller, G M Curr Opin Cell Biol 1995; 7:862-69).
Murine Embryonic Stem Cells
Study of vasculogenic events in murine ES cells has been instructive. Both hematopoietic and endothelial cells have been observed in blast cell colonies generated from mouse ES cell-derived embryoid bodies (Choi K, et al Development 1998; 125:725). Also working with murine ES cells, Nishikawa and colleagues demonstrated that 3-D embryoid body formation was not required for differentiation of lateral mesoderm cells. When cultured non-aggregated mouse embryonic cells were grown on a collagen substrate, cells expressing vascular endothelial Cadherin (VE-cad+) were found to give rise to hematopoietic cells (Nishikawa S I, et al Development 1998; 125:1747, Nishikawa S I et al Immunity 1998; 8:761, and Fujimoto T, et al Genes Cells 2001; 6:1113). Where markers of smooth muscle cell (SMC) phenotype (e.g. surface markers and morphology) are observed, early periendothelial SMCs associated with embryonic endothelial tubes can be shown to trans-differentiate from the endothelium (Gittenberger de-Groot, A C et al, Atheroscler Thromb Vasc Biol 1999; 19:1589), and differentiation of embryonic common vascular progenitors (Flk1+) into endothelial and smooth muscle cells can been observed (Yamashita J et al Nature 2000; 408:92). However, attempts to directly extrapolate from mouse to human EC systems have met with disappointing results, indicating that many developmental processes and requirements are species specific (see, for example, Reubinoff B E et al, Nat. Biotechnolog. 2000; 18:399-404). Specifically, in contrast to it's expression in mouse embryonic stem (mES) cells, the vascular specific growth factor receptor VEGFR 2 (Flk-1/KDR) is expressed in undifferentiated human embryonic stem cells (hES) (Kaufman, D S et al PNAS USA 2001; 98:10716-21) and does not increase during the first week of differentiation (Levenberg, S et al PNAS USA 2002; 99:4391-96), indicating that the timing of VEGFR 2 expression may vary among vertebrate species (also reviewed by Nishikawa; Nishikawa S I et al Curr Opin Cell Biol 2001; 13:862-69). Levenberg et al (Levenberg, S et al PNAS USA 2002; 99:4391-96) further reported that other endothelial markers, namely vascular endothelial cadherin (VE-cad) and platelet-endothelial cell adhesion molecule-1 (PECAM1/CD31), increased during the first week of hES differentiation. Clearly, coordination of expression of specific endothelial-specific factors, in the appropriate combinations, are crucial to human vasculogenesis.
Human Embryonic Stem Cells
Human embryonic stem (hES) cell lines were first derived in 1998 (Thomson, J A et al Science 1998; 282:1145; U.S. Pat. No. 6,200,806 to Thomson et al; U.S. Pat. No. 6,331,406 to Gearhart J D and Shamblott M J), and have recently been induced to differentiate in vitro in a cell lineage-specific manner (Schuldiner M et al PNAS 2000; 97:11307-312, International Patent Application WO0210347 A2 to Benvenisty, N). Since hES cells maintain the embryonic stem cell phenotype throughout hundreds of doubling times, and differentiate to all embryonic cell lineages, they provide a potentially unlimited source of cells for study and clinical application. Both hematopoietic and endothelial cell differentiation have been observed in human ES cells. To date, hematopoietic differentiation of the hES cells has required coculturing with either the S17 (murine bone marrow) or C166 (yolk sac endothelial) stromal cell lines, inducing the appearance of primary human hematopoietic tissue characteristics such as cell surface antigen CD34 and hematopoietic colony formation (Kaufman, D S et al PNAS USA 2001; 98:10716-21). In another recent study, endothelial cells were selected by cell sorting (FACS) from human embryoid bodies (EB) using monoclonal antibodies raised against the endothelial-specific marker PECAM-1 (Levenberg, S et al PNAS USA 2002; 99:4391-96). The selected, PECAM-1+ embryoid body-derived (EBD) cells exhibited endothelial-specific characteristics such as von Willebrand factor, VEGFR-2 and VE-cad surface markers and primitive, vessel-like cord formation when cultured on a soft substrate (Matrigel). PECAM-1+ EBD cells were further observed forming vascular structures in-vivo following seeding on biodegradable polymer matrix sponges and implantation into SCID mice. However, all of the abovementioned methods for differentiation of human. ES require either coculturing with non-human cells or embryoid body formation prior to appearance of endothelial phenotypes, and immunofluorescent cell sorting for selection according to endothelial cell markers, rendering them both costly and unsuitable for many clinical applications. Thus, it would be advantageous to provide a simplified, less expensive method of culturing, selecting and directing differentiation of human embryonic stem cells, without the limitations of aggregation into embryoid bodies or immunofluorescent selection.
Prior art discloses a number of techniques and methods for preparation and use of embryonic stem cells for differentiation. Early techniques required inner-cell mass cells from blastocyst-stage embryos (fresh or cryopreserved) as a source of stem cells (see, for example, International Patent Application No. WO 0129206 A1 to Cibelli et al; U.S. Pat. Application Publication Nos. 20020045259 A1 to Lim et al, 20020004240 A1 to Wang). Many others rely upon aggregation of the stem cells into embryoid bodies for initiation of differentiation (see, for example, International Patent Application No. WO 0070021 A3 to Itskovitz-Eldor J and Benvenisty N).
Various methods for differentiation of stem cells in culture have also been disclosed. International Patent Application No. WO 0134776 A1, U.S. Pat. Application Publication No. 20020015694 A1, and U.S. Pat. No. 6,280,718, all to Kaufman, D et al, disclose methods of differentiating human embryonic stem cells into hematopoietic cells by coculture with mammalian stromal cells. U.S. Patent Application Publication No. 20020023277 A1 to Stuhlmann, H et al discloses the identification and isolation of the vasculogenesis-related gene Vezf1 in mice, and methods for selection of endothelial cells and precursors based on Vezf1 expression. Also disclosed are methods for modulating angiogenesis, and diagnosis and treatment of vascular disease and neoplasm in a subject, the methods employing detection, measurement and modification of levels of Vezf1 in tissues. However, the transgenic ES cell experiments described were restricted to mouse embryoid body cells only, and neither human nor any other primate embryo cells were used. Furthermore, selection, according to the disclosure, is on the basis of Vezf1 expression, thus failing to overcome the abovementioned limitations of aggregation and immunofluorescent sorting.
U.S. Patent Application Publication No. 20020039724 A1 to Carpenter, M K discloses methods for differentiation and selection of human embryonic neural progenitor cells, and therapeutic, diagnostic and investigative uses thereof. The disclosed human neural progenitor cells, for reconstitutive therapy of, for example, neural degenerative disease, are also derived from human embryoid bodies, and are selected and isolated according to expression and detection of neural cell specific markers, NCAM and A2B5. Similarly, International Patent Application WO 0181549 A3 to Rambhatla L and Carpenter M K discloses methods for treating embryoid bodies with n-butyrate for induction of differentiation into hepatocyte lineage cells. No mention is made of non-aggregated hES origins, or simplified methods of progenitor isolation in either application.
Recently, Benevenisty (International Patent Application WO 0210347 A2 to Benvenisty) disclosed methods for “directed differentiation” of human embryonic stem cells by treating aggregated, embryoid body-derived cells with exogenous factors, enriching the cultures for a specific lineage cell type. The factors used were known effectors of differentiation, such as retinoic acid, neuronal growth factor, epidermal growth factor, fibroblast growth factor, etc., and differentiation was determined by de novo gene expression, and the appearance of tissue lineage-specific cell surface markers.
U.S. Pat. Application Publication No. 20010041668 A1, to Baron, M et al, discloses the use of extraembryonic, morphogenic gene products such as Hedgehog, TNF and WNT for modulation of hematopoiesis and vascular growth from mammalian adult and embryonic mesodermal-derived stem cells. Manipulation of the levels of these extra-embryonic gene products in the stem cell environment, via external application, or genetic engineering, for example, is disclosed for either enriching or diminishing the hematopoietic and/or vascular potential of stem cells for treatment and diagnosis of diseases involving blood abnormalities, hypervascularization, neovascularization and revascularization of tissues. However, although treatment of human embryonic tissues is proposed, no examples using human adult or embryonic cells are presented, and no methods for culture or selection of non-aggregated embryonic stem cells, designed to overcome the abovementioned limitations, are disclosed.
Thus, there exists a need for a simplified and inexpensive method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells. Such a method and the progenitor cells isolated thereby can be used for in-vitro vascular engineering, treatment of congenital and acquired vascular and hematological abnormalities, for evaluation and development of drugs affecting vasculo- and angiogenic processes, and for further investigation into tissue differentiation and development.